Monoubiquitination of FANCD2 at Lys561 during S phase is required for normal cell-cycle progression.
(A) PD20 (FA-D2) fibroblasts were transduced with pMMP-FANCD2 (wt), pMMP-FANCD2 (K561R), or pMMP (empty vector), and stably transduced cells were selected in puromycin. Indicated cells were synchronized by double-thymidine block, and whole-cell lysates from the indicated cell-cycle stages were immunoblotted with the anti-FANCD2 monoclonal antibody. (B) Indicated transfectants were synchronized at the G1/S boundary by double-thymidine block and synchronously released into S phase. Where indicated, the cells were exposed to MMC (500 nM) for 2 hours, at 1 to 3 hours after release, followed by the removal of MMC and the addition of fresh medium. Progression of the indicated cell lines from G2 to M phase was determined by quantitating the percentage of cells with an elevated FACS signal with the MPM-2 antibody, as previously described.30 31
